The anti-cancer drugs used to treat tumours are in most cases applied systemically and spread through the whole body of the patient. The high systemic dose of such drugs required for cancer treatment is combined with unpleasant side-effects for the patient.
In an attempt to circumvent this problem, cancer-prodrugs that have to be metabolized or activated in the body before they become cytotoxic have been used. Unfortunately, human tumours that contain appropriate high levels of the activating enzymes are rare. The main site for activation of prodrugs is the liver and to ensure that a tumour, at a distant site, receives a sufficient dose of the activated drug, the amount of activated prodrug produced in the liver has to be quite high and again this leads to toxic side effects for the patient.
One strategy by which these problems of high systemic concentration of activated drugs could be circumvented would be to provide means for activation of the prodrug directly in or near the site of the tumour. This strategy would require that tumour cells, or cells at the site of a tumour are genetically transformed to produce high amounts of the enzymes required for metabolizing the cancer prodrugs. Retroviral vectors are ideally suited for the stable delivery of genes to cells since the retrovirus is able to integrate the DNA form of its genome into the genome of the host cell and thus all daughter cells of an infected cell will carry the retroviral vector carrying the therapeutic gene. A further advantage is that most retroviruses only infect dividing cells and they are therefore ideal gene delivery vehicles for tumour cells.
Retroviral vectors are the most commonly used gene transfer vehicles for the clinical trials that have been undertaken to date. Most of these trials have, however, taken an ex vivo approach where the patient's cells have been isolated, modified in culture and then reintroduced into the patient.
The delivery of genes in vivo introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed.
A major concern for eventual in vivo gene therapy, both from a safety stand point and from a purely practical stand point, is the targeting of the expression. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important when the genes to be transferred are such prodrug activating genes designed to ablate specific tumour cells. Ablation of other, non-target cells would obviously be very undesirable.
The essentially random integration of the proviral form of the retroviral genome into the genome of infected cells has posed a serious ethical problem because such random integration may lead to activation of proto-oncogenes and thus lead to the development of a new cancer. Most researchers would agree that the probability of a replication defective retrovirus, such as all those currently used, integrating into or near a cellular gene involving in controlling cell proliferation is vanishingly small. However, it is generally also assumed that the explosive expansion of a population of replication competent retroviruses from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. It has however, been well documented that recombination events between components of the retroviral vector system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize this risk of recombination (Salmons, B. and Günzburg, W. H., Human Gene Therapy, 4(2):129-41 (1993).
Retroviral vector systems consist of two components:
    1) The retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.The second component is:    2) A cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with one or more plasmids carrying the genes enabling the modified retroviral vector to be packaged.
To generate a recombinant retroviral particle, the retroviral vector is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic gene is transcribed from the retroviral vector and packaged into the modified retroviral particles. These recombinant retroviral particles are then used to infect tumour cells during which the vector genome and any cytotoxic gene becomes integrated into the target cell's DNA. A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells but the DNA of the vector carrying the therapeutic is integrated in the cell's DNA and can now be expressed in the infected cell.
A number of retroviral vector systems have been previously described that should allow targeting of the carried cytotoxic genes (Salmons, B. and Gunzburg, W. H. Human Gene Therapy, 4(2):12941 (1993)). Most of these approaches involve either limiting the infection event to predefined cell types or using heterologous promoters to direct expression of linked heterologous therapeutic genes to specific tumour cell types. Heterologous promoters are used which should drive expression of linked genes only in the cell type in which this promoter is normally active or/and additionally controllable. These promoters have previously been inserted, in combination with the therapeutic gene, in the body of the retroviral vectors, in place of the gag, pol or env genes.
The retroviral Long Terminal Repeat (LTR) flanking these genes carries the retroviral promoter, which is generally non-specific in that it can drive expression in many different cell types (Majors, J. (1990), in “Retroviruses—Strategies of replication (Swanstrom, R. and Vogt, P. K., Eds.): Springer-Verlag, Berlin: 49-92). Promoter interference between the LTR promoter, and heterologous internal promoters, such as the tissue specific promoters, described above, has been reported. Additionally, it is known that retroviral LTR's harbour strong enhancers that can, either independently, or in conjunction with the retroviral promoter, influence expression of cellular genes near the site of integration of the retrovirus. This mechanism has been shown to contribute to tumourigenicity in animals (van Lohuizen, M. and Berns, A. (1990), Biochim. Biophys. Acta, 1032:213-235). These two observations have encouraged the development of Self-Inactivating-Vectors (SIN) in which retroviral promoters are functionally inactivated in the target cell (WO 94/29437). Further modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double copy vectors (WO 89/11539). However, in both these vectors the heterologous promoters inserted either in the body of the vector, or in the LTR region are directly linked to the therapeutic gene.
The previously described SIN vector mentioned above carrying a deleted 3′LTR (WO 94/29437) utilizes in addition a heterologous promoter such as that of Cytomegalovirus (CMV), instead of the retroviral 5′LTR promoter (U3-free 5′LTR) to drive expression of the vector construct in the packaging cell line. A heterologous polyadenylation signal is also included in the 3′LTR (WO 94/29437).
A variety of cytotoxic genes carried by retroviral vectors have already been tested. These genes encode enzymes which convert substances that are pharmacodynamically and toxicologically inert even at high dose-levels but which can be converted in vivo to highly active metabolites (Connors, T. A., Gene Therapy, 2:702-709 (1995)).
In cancer chemotherapy appropriately designed prodrugs have been found to be effective in the treatment of animal tumours possessing high levels of an activating enzyme (Connors, T. and Whisson, M., Nature, 210:866 867 (1966); Cobb, L. et al., Biochemical Pharmacology, 18:1519-1527 (1969)). Clinical results were, however, disappointing since it was found that human cancers that contained appropriately high levels of activating enzymes were rare (Connors, T., Xenobiotica, 16:975-988 (1986)). Viral directed enzyme prodrug therapy (VDEPT) and the more general gene directed enzyme prodrug therapy (GDEPT) are related in that they also aim to destroy tumour cells by the tumour specific activation of a prodrug. However, in this case, the gene encoding the enzyme is either specifically targeted to malignant cells or is under the control of a specific promoter.
Up to now most of the efforts directed towards prodrug therapy have concentrated on the use of the human Herpes Simplex Virus thymidine kinase (HSV-tk) as a suicide gene. Although the HSV-tk enzyme in combination with the prodrug ganciclovir (GCV) has been recommended as a good system for GDEPT (Culver, K. et al., Science, 256:1550-1552 (1992); Ram, Z. et al., Cancer Research 53:83-88 (1993); Chen, S. Shine, H. et al., Proc. Natl. Acad. Sci., 91:3054-3057 (1994)) there are a number of theoretical considerations that would suggest that it is by no means the best combination. First, it is an S-phase specific agent with no effect on resting cells. This is because the GCV monophosphate is short lived and has to be present when cells are entering the S-phase to give a toxic effect. The HSV-tk phosphorylates GCV to the monophosphate form (a reaction that cannot be performed by mammalian enzymes) which is then phosphorylated by cellular enzymes to the triphosphate form and incorporated into DNA. Second, the active drug is a triphosphate and would not be expected to diffuse freely to cause a bystander effect. However a bystander effect has been observed both in vitro and in vivo although metabolic cooperation appears to be involved and in the latter case some of the effect may be an indirect one involving an immune component (Bi, W., Parysek, L. et al., Human Gene Therapy, 4:725-731 (1993); Vile, R. and Hart, I., Cancer Research, 53:3860-3864 (1993) and Freeman, S., Abboud, C. et al., Cancer Research, 53:5274-5283 (1993)). One disadvantage is that the bystander effect is dependent on a cell—cell contact. This may be due to the presence of gap junctions formed by intimate contact between the transduced and the surrounding cells which enable the transfer of phosphorylated ganciclovir.
Recently, interesting results have been reported with cells that have been transfected with the gene encoding the rat cytochrome P450 form 2B1 and then treated with cyclophosphamide (Chen, S., Shine, H et al., Proc. Natl. Acad. Sci., 91:3054-3057 (1994)).
Cytochrome P450's form a broad group of mono-oxygenases that catalyze oxidation of a wide range of substrates. They are produced by some bacteria, yeast, and by higher organisms, where they play a role in detoxification of xenobiotics, bioactivation reactions, and metabolism of various endogenous compounds.
Cytochrome P450 catalyzes the hydroxylation of the commonly used cancer prodrugs cyclophosphamide (CPA) and ifosfamide to their active toxic forms. Normally the expression of the patient's endogenous cytochrome P450 gene is limited to the liver, and anti-tumour effects of systemically applied CPA depends upon the subsequent systemic distribution of toxic drug metabolites from the liver. This has led to toxicity problems since the activated drug not only affects the tumour but also affects other normal patient tissues such as bone marrow and kidney.
A therapeutic approach to overcome said systemic toxicity problems would be a direct delivery of the activated metabolites. Unfortunately said metabolites have after in vitro production a short half life of about 30 min. (Sladek, N. E., Powers, J. F. & Grage, G. M., Half-life of oxazaphosphorines in biological fluids. Drug Metab. Dispos., 12, 553-559 (1984)).
Thus, in an alternative therapeutic approach, as addressed in PCT/US95/10365, the cytochrome P450 gene is selectively introduced directly into tumour cells, and overexpressed in these cells. Toxic metabolites produced from the transduced cells affect surrounding non-transduced tumour cells in a concentration gradient dependent manner. An additional advantage of the cytochrome P-450/CPA system is the lack of dependency upon cell replication for cytotoxic effects on the surrounding cells. This is because one of the active metabolites generated causes interstrand crosslinks regardless of the cell cycle phase. Later on, during DNA synthesis, these interstrand crosslinks result in cell death.
For the treatment of cancers, it would be feasible to isolate cells from a patient (either tumour cells or normal cells) infect them in vitro with a recombinant retroviral particle carrying a gene encoding cytochrome P450, and then return them to the patient in the vicinity of the tumour. However, this approach is extremely labor intensive because each patient cells must be isolated, cultured, transduced with the gene construct and successfully returned without infection by adventitious agents. The cost and time involved in such an approach limits its practical usefulness. Moreover, most tumours are not suitable for ex vivo gene therapy.
Ideally, the gene encoding cytochrome P450 should be introduced in vivo into the tumour cells, or into cells in the vicinity of the tumour. PCT/US95/10365 suggests an in vivo infection of such cells with isolated retroviral particles. Unfortunately, retroviral particles have a very short half life in vivo and additionally, said particles are very quickly cleared by the immune system. Thus, the infection efficiency in normal tumours and thereby the expression of the prodrug activating enzyme in tumour cells is very poor.
In a further set-up of PCT/US95/10365 cytochrome P450 producing retroviral packaging cells were injected into the brain to provide the retroviral particles and additionally the activating enzyme at the site of a tumour. Even if, using this approach, the amount of activating enzyme could be increased, compared to the efficiency of only an infection with retroviral particles, it nevertheless has the drawback that this approach is clearly limited to the brain, since only the less active immune system in the brain would tolerate the injection of packaging cells, which is derived from a different organism.
Thus, it would be highly desirable if an approach could be envisaged, where one type of cells is transfected with the gene encoding P450 or infected with a recombinant retroviral particle carrying a gene encoding P450, and then used for therapy of many different patients as well as for many different tumours. Such an approach is much more feasible, assuming that problems of immune rejection can be overcome without weakening the patients immune status.